Differentially expressed genes related to lymph node metastasis in advanced laryngeal squamous cell cancers.

Understanding the molecular mechanisms and gene expression in laryngeal squamous cell carcinoma (LSCC) may explain its aggressive biological behavior and regional metastasis pathways. In the present study, patients with locally advanced LSCC tumors were examined for differential gene expression in the normal mucosa (non-tumoral mucosa), tumors and lymph node tissues. The aim was to identify possible predictive genes for lymph node metastasis. A total of 16 patients who had undergone total laryngectomy with neck dissection for advanced LSCC were randomly selected from a hospital database: Eight of the patients had lymph node metastasis (Group 1) and the other eight patients did not have metastasis (Group 2). Overall survival (OS), disease-free survival (DFS) and disease-specific survival (DSS) were analyzed. For each patient, paraffin-embedded tissue samples were collected from non-tumoral mucosa, tumoral lesions and lymph node tissues. RNA was extracted from the tissue samples and used for complementary DNA synthesis, and microarray analysis was subsequently performed on each sample. Gene expression levels were determined in each specimen, and Groups 1 and 2 were compared and statistically analyzed. The microarray results for lymph node metastasis-positive and -negative groups, indicated the differential expression of 312 genes in the lymph nodes, 691 genes in the normal mucosal tissue and 93 genes in the tumor tissue. Transgelin (TAGLN) and cofilin 1 (CFL1) were identified as possible target genes and validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR results for TAGLN and CFL1 supported the microarray data. OS, DFS and DSS times were longer in Group 2 than in Group 1 (P=0.002, 0.015 and 0.009, respectively). In addition, TAGLN and CFL1 were associated with DFS and DSS. On the basis of these results, it is suggested that TAGLN and CFL1 expression may play an important role in the pathogenesis of regional metastasis and poor prognosis in advanced LSCC.

Neonatal Bladder-derived Mesenchymal Stem Cells Ameliorate Bladder Dysfunction in Diabetic Rat Models.

PURPOSE: To evaluate the effect of a new mesenchymal stem cell type derived from the neonatal bladder (nMSC-B) on diabetic bladder dysfunction (DBD). MATERIALS AND METHODS: nMSC-B were harvested from neonatal male Sprague-Dawley rat's bladder and expanded in culture. nMSC-B were transferred to Type-1 diabetic rats which were induced by a single dose 45 mg/kg Streptozocin (STZ). Stem cells were transferred via intraperitoneally (IP) (DM-IP group, n:6) and by direct injection to the detrusor (DM-D group, n:6) at 12th week following diabetes and compared with Phosphate Buffered Saline (PBS) injected diabetic rats (DM-PBS group, n:6) and age-matched PBS injected non-diabetic normal rats (NR-PBS group, n:6). All rats were evaluated histopathologically and functionally four weeks after the stem cell treatment. RESULTS: nMSC-B showed improvement in both voiding function and bladder structure. The maximum voiding pressure (MVP) values in the DM-PBS group were lower compare to DM-IP, DM-D and NR-PBS groups (13.27 ± 0.78 vs 16.27 ± 0.61, 28.59 ± 2.09, 21.54 ± 1.00, respectively, P < .001). There was a significant improvement for MVP values in stem cell-treated groups. Immunohistochemical examination revealed decreased bladder smooth muscle (SM), increased fibrosis and desquamation in urothelia in diabetic groups compared to normal group(P < .001). We detected recovery in the stem cell groups. This recovery was more evident in DM-D group.  No statistical difference was observed in SM and fibrosis between DM-D and NR-PBS groups (P = .9). CONCLUSION: It was shown that nMSCBs provided amelioration of DBD. We think that nMSC-B constitutes an effective treatment method in DBD.

Neonatal Bladder-derived Mesenchymal Stem Cells Ameliorate Bladder Dysfunction in Diabetic Rat Models.

PURPOSE: To evaluate the effect of a new mesenchymal stem cell type derived from the neonatal bladder (nMSC-B) on diabetic bladder dysfunction (DBD). MATERIALS AND METHODS: nMSC-B were harvested from neonatal male Sprague-Dawley rat's bladder and expanded in culture. nMSC-B were transferred to Type-1 diabetic rats which were induced by a single dose 45 mg/kg Streptozocin (STZ). Stem cells were transferred via intraperitoneally (IP) (DM-IP group, n:6) and by direct injection to the detrusor (DM-D group, n:6) at 12th week following diabetes and compared with Phosphate Buffered Saline (PBS) injected diabetic rats (DM-PBS group, n:6) and age-matched PBS injected non-diabetic normal rats (NR-PBS group, n:6). All rats were evaluated histopathologically and functionally four weeks after the stem cell treatment. RESULTS: nMSC-B showed improvement in both voiding function and bladder structure. The maximum voiding pressure (MVP) values in the DM-PBS group were lower compare to DM-IP, DM-D and NR-PBS groups (13.27 ± 0.78 vs 16.27 ± 0.61, 28.59 ± 2.09, 21.54 ± 1.00, respectively, P < .001). There was a significant improvement for MVP values in stem cell-treated groups. Immunohistochemical examination revealed decreased bladder smooth muscle (SM), increased fibrosis and desquamation in urothelia in diabetic groups compared to normal group(P < .001). We detected recovery in the stem cell groups. This recovery was more evident in DM-D group.  No statistical difference was observed in SM and fibrosis between DM-D and NR-PBS groups (P = .9). CONCLUSION: It was shown that nMSCBs provided amelioration of DBD. We think that nMSC-B constitutes an effective treatment method in DBD.

Preparation and characterization of novel albumin-sericin nanoparticles as siRNA delivery vehicle for laryngeal cancer treatment.

Small interfering RNA (siRNA)-based gene silencing strategy has high potential on suppressing specific molecular targets, involved in cancer progression. However, the lack of an effective nanocarrier system that safely delivers siRNA to its target still limits the clinical applications of siRNA. This study aimed to develop albumin-sericin nanoparticles (Alb-Ser NPs) as a novel siRNA delivery system for laryngeal cancer treatment. Nanoparticle formulations composed of albumin and sericin at different ratios (1:1, 2:1, 1:2 w/w) were synthesized by desolvation method. The nanoparticles were modified with poly-L-lysine (PLL) for siRNA binding and decorated with hyaluronic acid (HA) to target laryngeal cancer cell line, Hep-2. HA/PLL/Alb-Ser NPs were individually loaded with siRNAs for casein kinase 2 (CK2), Absent, Small, or Homeotic-Like (ASH2L), and Cyclin D1 genes, which are overexpressed in Hep-2 cells. Downregulation of genes was confirmed by real-time PCR (RT-PCR). Size, morphological, and thermogravimetric characterizations revealed that Alb-Ser NPs having 2:1 (w/w) ratio are the most optimized formulation. Between 36.8 and 61.3% of siRNA entrapment efficiencies were achieved. HA/PLL-siRNA/Alb-Ser (2:1) NPs-mediated gene silencing resulted in a significant inhibition of cell growth and induction of apoptosis in cells. Our findings showed that HA/PLL/Alb-Ser (2:1) NPs were promising as a siRNA carrier.

Determination of the effects of bone marrow derived mesenchymal stem cells and ovarian stromal stem cells on follicular maturation in cyclophosphamide induced ovarian failure in rats.

OBJECTIVE: Chemotherapy causes depletion of primordial follicles that leads to premature ovarian failure in female cancer survivals. We investigated the effect of bone marrow derived mesenchymal (BMMSCs) and ovarian stromal stem cells (OSSCs) on follicle maturation in chemotherapy induced ovarian failure. MATERIAL AND METHODS: Thirty six Wistar Albino female rats were divided into three groups. Cyclophosphamide at a dose of 200 mg/kg was intraperitoneally (IP) given to the rats in all groups two times. 4 × 106 BMMSCs (IP) was injected to the group-2 and 4 × 106 OSSCs (IP) was injected to the group-3. Serum Anti-Müllerian Hormone (AMH) levels was determined with ELISA and primordial follicles were counted for investigation of primordial follicle reserve. The ovarian structure were evaluated histomorphologically. Localization of BrdU labeled stem cells, the expression of the cell cycle regulator p34Cdc2, gap junction protein p-connexin43 and intraovarian regulators of folliculogenesis Bone Morphogenic Protein 6 and 15 (BMP-6 and BMP-15) were investigated by immunohistochemistry. RESULTS: The immunstaining of BMP-6 was higher in oocytes of group-3 more than group-1 and group-2. The immunpositivity of p34cdc2 and BMP-15 were also higher in follicular cells of group-3 than the other groups. The presence of p-connexin43 in group-3 was determined more than group-1 and group-2. The ovarian follicles with normal histological structure were observed just in group-3. Although, The AMH levels were decreased in rats from all groups at the end of experimental procedure the primordial follicle counts in group-3 was significantly higher than group-1. CONCLUSION: Our findings suggest that OSSCs have more protective effect on follicle maturation than BMMSCs in cyclophosphamide induced ovarian damage.

Epigenetic silencing of the tumor suppressor genes SPI1, PRDX2, KLF4, DLEC1, and DAPK1 in childhood and adolescent lymphomas.

The aim of the study was to investigate the expression and methylation status of seven distinctive genes with tumor suppressing properties in childhood and adolescent lymphomas. A total of 96 patients with Hodgkin Lymphoma (HL, n = 41), Non-Hodgkin Lymphoma (NHL, n = 15), and reactive lymphoid hyperplasia (RLH, n = 40, as controls) are included in the research. The expression status of CDKN2A, SPI1, PRDX2, DLEC1, FOXO1, KLF4 and DAPK1 genes were measured with QPCR method after the RNA isolation from paraffin blocks of tumor tissue and cDNA conversion. DNA isolation was performed from samples with low gene expression followed by methylation PCR study specific to promoter regions of these genes. We found that SPI1, PRDX2, DLEC1, KLF4, and DAPK1 genes are significantly less expressed in patient than the control group (p = 0.0001). However, expression of CDKNA2 and FOXO1 genes in the patient and control groups were not statistically different. The methylation ratios of all genes excluding the CDKN2A and FOXO1 were significantly higher in the HL and NHL groups than the controls (p = 0.0001). We showed that SPI1, PRDX2, DLEC1, KLF4 and DAPK1 genes are epigenetically silenced via hypermethylation in the tumor tissues of children with HL and NHL. As CDKN2A gene was not expressed in both patient and control groups, we conclude that it is not specific to malignancy. As FOXO1 gene was similarly expressed in both groups, its relationship with malignancy could not be established. The epigenetically silenced genes may be candidates for biomarkers or therapeutic targets in childhood and adolescent lymphomas.

The effects of the stem cell on ciliary regeneration of injured rabbit sinonasal epithelium.

Defects in mucosal healing after sinonasal surgery cause infection, scar formation causing obstruction, relapse of the disease within a shorter period and revision surgery. The present study aimed to create a functional ciliated epithelium using a stem cell and stem cell sheet of adipose tissue origin and to show such regeneration ultra-structurally on experimentally injured rabbit nasal epithelium. This was an experimental animal study and basic research. A total of 18 white New Zealand rabbits were divided into three groups. The medial wall of the maxillary sinus of the subjects was peeled off bilaterally. No additional procedure was applied to the subjects in Group 1. In Group 2, adipose tissue-derived mesenchymal stem cell was implanted on the wound edges of the subjects. In Group 3, a stem cell sheet of three layers was laid onto the defect area. All subjects were killed after 3 weeks. The presence of the stem cell stained with bromo-deoxyuridine was assessed with a light microscope, whereas cilia density, ciliated orientation and cilia structure were evaluated with a scanning electron microscope. Ciliary densities in Group 2 and Group 3 were statistically superior compared to the control group (p < 0.001, p = 0.007). Cilia morphology in Group 2 and Group 3 was also better than the control group (p < 0.01, p = 0.048). Ciliary orientation in Group 2 was scored highest (p < 0.01). The ratio of BrDu-stained cells was observed to be 27% in Group 3 and 8% in Group 2. Sub-epithelial recovery was observed to be better in Group 3. Adipose tissue-derived mesenchymal stem cell increased the healing of the injured maxillary sinus mucosa of the rabbits in terms of cilia presence, density and morphology regardless of the implementation technique. Level of evidence NA.

The effects of commonly used intravitreal steroids on proliferation index of ciliary body-derived mesenchymal stem cells: an in vitro study.

AIM: To investigate the effects of commonly used intravitreal steroids on survival and proliferation (namely, proliferation index) of ciliary body-derived mesenchymal stem cells (CB-MSC). METHODS: CB-MSCs were isolated from newborn rats' eye, and they were expanded in the medium. Commonly used intravitreal steroids such as dexamethasone (Dex) and triamcinolone acetonide (TA) were added into the medium at commonly used concentration in clinical practice (0.1 mg/mL) and at lower concentration (0.01 mg/mL). Proliferation indexes of CB-MSCs were analyzed with the xCELLigence system at nine consecutive times (at 3rd, 6th, 21th, 30th, 45th, 60th, 75th, 90th and 100th h). RESULTS: Both TA and Dex at both 0.01 mg/mL and 0.1 mg/mL concentrations had negative effect on proliferation indexes of CB-MSC. Although negative effect of TA on proliferation index of CB-MSC at both concentrations was not statistically significant, statistically significant negative effect of Dex at 0.01 mg/mL concentration started 60th h (p = 0.017) and 0.1 mg/mL concentration started 30th h (p = 0.014). DISCUSSION: Even therapeutic doses of intravitreal corticosteroid agents might have negative effects on limited numbers of stem cells. Especially, Dex caused statistically significant toxic effects on CB-MSCs even at lower concentrations of those used clinically. These novel findings deserve further in vivo investigations.

Nonclassical Congenital Adrenal Hyperplasia and Pregnancy.

Objective. The most common form of congenital adrenal hyperplasia (CAH) is 21-hydroxylase (21-OH) deficiency due to mutation of the CYP21A2 gene. Patients with nonclassical CAH (NC-CAH) are usually asymptomatic at birth and typically present in late childhood, adolescence, or adulthood with symptoms of excessive androgen secretion. Subfertility is relative in NC-CAH, but the incidence of spontaneous miscarriage is higher. Here, we report a previously undiagnosed female who gave birth to a normal male child and is planning to become pregnant again. Case Report. A 32-year-old female was referred to our clinic for obesity. Her medical history revealed that she had had three pregnancies. She was planning to become pregnant again. Her laboratory results revealed that she had NC-CAH. Since her husband is the son of her aunt and she had miscarriages and intrauterin exitus in her history, their genetic analyses were performed. Conclusion. Since most patients with NC-CAH have a severe mutation, these patients may give birth to a child with the classical CAH (C-CAH) if their partner is also carrying a severe mutation. Females with NC-CAH who desire pregnancy must be aware of the risk of having an infant with C-CAH.

Angiotensin converting enzyme and methylenetetrahydrofolate reductase gene variations in fibromyalgia syndrome.

OBJECTIVE: Fibromyalgia syndrome (FM) is a common disease characterized by generalized body pain, sensitivity in certain physical areas (sensitive points), lowered pain threshold, sleep disorder, and fatigue. The study aimed to determine the effects ACE I/D and MTHFR C677T gene polymorphisms in Turkish patients with FM and evaluate if there was an association with clinical features. METHODS: This study included 200 FM patients and 190 healthy controls recruited from the department of Physical Medicine and Rehabilitation at Gaziosmanpasa University in Tokat, Turkey. ACE I/D polymorphism genotypes were determined by using polymerase chain reaction (PCR) by specific primers. The MTHFR C677T mutation was analyzed by PCR-based restriction fragment length polymorphism (RFLP) methods. RESULTS: We found a statistically significant relation between ACE polymorphism and FM (p<0.001, OR: 1.71, 95% CI: 1.28-2.27). However, this was not the case for ACE polymorphism and the clinical characteristics of the disease. There was also no statistically significant relation between MTHFR C677T mutation and FMS (p>0.05, OR: 1.20, 95% CI: 0.82-1.78), but dry eye and feeling of stiffness which are among the clinical characteristics of FMS were significantly related with MTHFR C677T mutation (p<0.05). CONCLUSION: Our findings showed that there are associations of ACE I/D polymorphism with susceptibility of a person for development of fibromyalgia syndrome. Also, it is determined an association between MTHFR C677T polymorphism and feeling of stiffness and dry eye which are among the clinical characteristics of FM. Our study is the first report of ACE I/D and MTHFR C677T polymorphisms in fibromyalgia syndrome.

Effect of allogeneic limbal mesenchymal stem cell therapy in corneal healing: role of administration route.

AIMS: To investigate whether allogeneic limbal mesenchymal stem cell (LMSC) therapy affects corneal healing after a severe chemical burn and whether the route of administration of LMSCs differs in its therapeutic effect in this respect. METHODS: A total of 60 Sprague-Dawley rats with clinically proven alkali injury were divided into four equal groups (n = 15) as follows: group 1: 2 × 10(5) cells/drop LMSCs, topically applied 6 times a day for 2 days; group 2: 2.4 × 10(6) cells in 0.5 ml LMSCs, subconjunctivally applied; group 3: 2.4 × 10(6) cells in 1 ml LMSCs, intraperitoneally applied, and group 4: no LMSC treatment. The groups were compared according to grades of corneal opacity (CO), corneal neovascularization (CNV) and corneal fluorescein staining (CFS). The migration of LMSCs into the cornea and the inflammatory characteristics of the groups were evaluated with BrdU (5-bromo-2'-deoxyuridine bromodeoxyuridine) immunostaining and histopathologically in a 4-week follow-up. RESULTS: There were statistically significant differences between the LMSC-treated and control groups in each week regarding mean CO scores and in the 3rd week regarding the mean CNV and CFS scores (p < 0.05). The statistical significance was due to the differences between the topical and the control group and between the subconjunctival and the control group. BrdU+ LMSCs were seen in the corneal epithelium of the all LMSC-administered rats, and fewer inflammatory changes were observed in these rats. CONCLUSION: Allogeneic LMSC treatment, especially topical and subconjunctival administration, seems to be helpful in affecting corneal healing after a severe corneal burn.

Efficacy of topical mesenchymal stem cell therapy in the treatment of experimental dry eye syndrome model.

Purpose. The current study was set out to address the therapeutic efficacy of topically applied mesenchymal stem cells (MSCs) on dry eye syndrome (DES) induced by benzalkonium chloride (BAC) in rats. Methods. Rats were divided into two groups just after establishment of DES. Eye drops containing either bromodeoxyuridine labeled MSCs (n = 9) or phosphate buffer solution (n = 7) were topically applied once daily for one week. Schirmer test, break-up time score, ocular surface evaluation tests, and corneal inflammatory index scoring tests were applied to all rats at baseline and after treatment. All rats were sacrificed after one week for histological and electron microscopic analysis. Results. Mean aqueous tear volume and tear film stability were significantly increased in rats treated with MSCs (P < 0.05). Infiltration of bromodeoxyuridine labeled MSCs into the meibomian glands and conjunctival epithelium was observed in MSCs treated rats. Increased number of secretory granules and number of goblet cells were observed in MSCs treated rats. Conclusion. Topical application of MSCs could be a safe and effective method for the treatment of DES and could potentially be used for further clinical research studies.

Keratinocyte growth factor-2 and autologous serum potentiate the regenerative effect of mesenchymal stem cells in cornea damage in rats.

AIM: To investigate the healing process after severe corneal epithelial damage in rats treated with mesenchymal stem cells (MSCs) cultured with or without keratinocyte growth factor (KGF-2) and autologous serum (AS) on amniotic membrane (AM). Many patients are blind and devastated by severe ocular surface diseases due to limbal stem cell deficiency. Bone marrow-derived MSCs are potential sources for cell-based tissue engineering to repair or replace the corneal tissue, having the potential to differentiate to epithelial cells. METHODS: The study included 5 groups each including 10 female "Sprague Dawley" rats in addition to 20 male rats used as bone marrow donors. Group I rats received AM+MSCs, Group II rats AM+MSCs cultured with KGF-2, Group III rats AM+MSCs cultured with KGF-2+AS, Group IV rats only AM and Group V rats, none. AS was derived from blood drawn from male rats and bone marrow was obtained from the femur and tibia bones of the same animals. Therapeutic effect was evaluated with clinical, histopathological and immunohistochemical assessment. MSC engraftment was demonstrated via detection of donor genotype (Y+) in the recipient tissue (X) with polymerase chain reaction. RESULTS: Corneal healing was significantly better in Groups I-III rats treated with MSC transplantation compared to Group IV and Group V rats with supportive treatment only. The best results were obtained in Group III rats with 90% transparency, 70% lack of neovascularization, and 100% epithelium damage limited to less than 1/4 of cornea. CONCLUSION: We suggest that culture of MSCs with KGF-2 and AS on AM is effective in corneal repair in case of irreversible damage to limbal stem cells.

The contralateral extremity has also benefit from the locally administered bone marrow-derived mononuclear cells and cord blood serum in diabetic ischemic wound healing.

OBJECTIVE: Impaired wound healing could be a disaster especially in diabetes and amputation is the major risk. The aim of this study was to evaluate the benefit of BMMCs and CBS on wound healing. METHODS: Diabetic rats were underwent bilateral limb ischemia and wounding of skin defects on both extremities. The groups were formed as BMMCs (group A), BMMCs and CBS (group B), only CBS (group C), and phosphate buffer solution (group D) that were injected into wounds on right legs. RESULTS: The complete recovery of right legs was established as a mean of 21.4 ± 1.1 days, 12.9 ± 1.5 days, 30.0 ± 0.0 days and 38.1 ± 1.5 days according to Groups A, B, C, and D (p < 0.05). The recovery of left legs were calculated as a mean of 27.0 ± 0.0 days, 24.0 ± 0.0 days, 35.6 ± 1.1 days and 37.3 ± 1.6 days according to Groups A, B, C and D (p < 0.05). At the end of the recovery, the HE staining showed that vascularity was increased in groups A and B. CONCLUSION: Transplantation of BMMCs and CBS to the ischemic wounds of the diabetic rats accelerate the repair. The recovery was also superior in the same group although the treatment was not applied to the left extremity directly.

Resveratrol and adipose-derived mesenchymal stem cells are effective in the prevention and treatment of doxorubicin cardiotoxicity in rats.

Anthracyclines can cause severe cardiac toxicity leading to heart failure. The aim of this study was to determine the effects of cardioprotective polyphenolic compound resveratrol (RES) and adipose-derived mesenchymal stem cells (ADMSCs) on cardiac tissue of rats treated with doxorubicin (DOX). Forty-two female and three male Wistar-Albino rats were included in the study. The study groups and the control groups were as follows: Group I: DOX; Group II: DOX + RES; Group III: DOX + ADMSCs; Group IV: DOX + RES + ADMSCs; Group V: Sham operation; and Group VI: normal saline. ADMSCs obtained from male rats were defined with stem cell markers [CD11b/c(-), CD45(-), CD90(+), CD44(+), and CD49(+)]. DOX 12 mg/kg intraperitoneally (i.p.) was injected as a single dose in female rats. Resveratrol 100 mg/kg was injected three times i.p. in Groups II and IV. ADMSCs 2 × 10(6) cells/kg/dose were labeled with bromodeoxyuridine (BrdU) and injected i.p. for a total of three times in Groups III and IV. When the study was terminated after 4 weeks, the beating hearts were connected to a Langendorff setup and records were obtained for 30 minutes. Histopathological, immunhistochemical, and immunofluorescent examination with H&E, Troponin I, and BrdU stains were also performed. Also, ADMSCs were demonstrated in the myocardium of transplanted rats. Left ventricle functions and myocardial histology demonstrated significant impairment in DOX only group compared to groups with ADMSCs (P < .05). We suggest that RES and ADMSCs were successful in the prevention and treatment of the doxorubicin cardiomyopathy in rats. The hypothetical mechanisms of regeneration are multiple, including cell differentiation and autocrine/paracrine effects of ADMSCs.